Transgenic animals

1. Transgenic Animals,Transgenic Animals,
Knock-Out Mice andKnock-Out Mice and
DollyDolly

2. Transgenic Animals: A Focus
on Transgenic Mice Studies
http://www.hku.hk/biochem/tgcentre/transcentre.html

3. Introduction
 Transgenic animals:
– Animals which have been genetically engineered to
contain one or more genes from an exogenous source.
– Transgenes are integrated into the genome.
– Transgenes can be transmitted through the germline to
progeny.
– First transgenic animal produced = “Founder Animal”

4. Introduction of foreign genes
into intact organisms
 Procedure is basically the same regardless of
which animal is involved.
 Integration usually occurs prior to DNA
replication in the fertilized oocyte.
– Majority of transgenic animals carry the gene in all of
their cells, including the germ cells. Transmission to
next generation requires germline integration.
– Some integration events occur subsequent to DNA
replication giving rise to mosaic animals which may
or may not contain the transgene in its germline.

5. Procedure for Producing
Transgenic Mice
 Three different breeding pairs of mice are
required.

6.  First Breeding Pair:
– Fertile male + superovulated female
• Fertile male = stud (changed regularly to ensure
performance)
• Superovulated female = immature female induced to
superovulate
– Pregnant mare’s serum (=FSH) on day 1
– Human Chorionic Gonadotropin (=LH) on day 3
• Mated on day 3
• Fertilized oocytes microinjected on day 4 with foreign DNA
construct.
• Microinjected oocytes are transferred to the oviducts of
surrogate mothers at end of day 4.

7.  Second breeding pair:
– Sterile male + surrogate mother
• Sterile male produced through vasectomy
• Surrogate mother must mate to be suitable recipient of
injected eggs
• Mated on day 3
• Microinjected oocytes from first breeding pair are
transferred to oviducts on day 4
• Embryos implant in uterine wall and are born 19 days later.
• Southern blotting techniques confirm presence and copy
number of transgenes.

8.  http://www.itba.mi.cn
r.it/human_g...transge
nic.html

9.  Third breeding pair:
– Foster parents
• Fertile male + female mated to give birth on same
day surrogate mother
• Serves as foster parent if caesarian section is
required for surrogate mother

11. Manipulation ofManipulation of
Fertilized OocytesFertilized Oocytes
Refer to transparencies
and slides presented in
class.

13. Flow Cytometry for the Analysis of Transgenic Mice

15. Integration of Transgene into
One Chromosome
 Normally the transgene inserts into one
chromosome giving rise to a heterozygote.
– 50% probability of passing transgene onto offspring.
 Two heterozygous mice may be bred to obtain a
homozygous line that contains the transgene on
both chromosomes.
– 100% probability of passing transgene onto offspring.
 Most transgenes are stably transmitted for many
generations without detectable rearrangement.

16. Mechanisms of DNA
Integration
 Linear molecules integrate more efficiently than circular
molecules (~5x)
 Once in the oocyte, the linear molecules circularize.
 Usually all of the molecules that integrate are on the same
chromosome and at the same site.
 Multiple copies are usually arranged in a tandem, head-
to-tail array.
 The size of the DNA molecule (0.7 – 50Kb) is not an
important parameter.
 The concentration and purity of the injected DNA is
critical (1-3 µg/ml maximum).

17. Working Hypothesis of DNA
Integration
 The ends of the injected linear DNA integrate at breaks that occur
spontaneously in the chromosome.
 Other injected molecules which have circularized probably
recombine with each other and the integrated copies to generate a
tandem, head-to-tail array.
 Recombination is probably favored because of high local DNA
concentration and special properties such as the absence of normal
chromatin structure.
 The number of chromosomal breaks is presumably limiting
explaining the low number of integration events and why different
DNA molecules are usually integrated at the same site.

18. Gene Expression in
Transgenic Mice
 In order to discriminate the products of the
injected gene from those of an endogenous
counterpart, the injected gene must be marked in
some way.
– Mini-genes where exons are deleted of cDNA where
introns are absent.
– Modification by insertion/deletion/mutagenesis of a
few nucleotides (e.g. the gain or loss of a restriction
endonuclease site).
– Hybrid genes where foreign epitopes are expressed on
transgenic products.

19. Tissue-Specific Gene
Expression
 Generally, if a tissue-specific gene is expressed at all,
then it is expressed appropriately, despite the fact that it
has integrated at a different chromosomal location.
– Trans-acting proteins involved in establishing tissue-specific
expression are capable of finding their cognate sequences and
activation transcription at various chromosomal locations.
– Levels of expression vary between founder animals as
chromosomal position can influence accessibility of the
transgenes to these trans-acting transcription factors.
– Some founders do not express the transgene at all owing to
integration into heterochromatin domains where DNA is
methylated heavily (silent).

20. Prokaryotic Sequences Must be
Removed for Optimal Expression
 Prokaryotic sequences derived from the plasmid or
bacteriophage vector used for replication of the transgene
in bacteria can be inhibitory or “poisonous” for some
transgenes.
 Therefore, the transgene fragment requires purification
from contaminating vector sequence prior to
microinjection.

21. Possible Reasons for Lack of
Transgene Expression
 Integration in cis-acting silencer sequences (the negative counterpart
of enhancer elements) might be sites for covalent modification of
DNA (e.g. methylation) which might initiate condensation into an
inactive chromatin configuration, or they might phase nucleosomes
in an inappropriate manner.
 The inadvertent loss of certain regulatory sequences during the
production of the constructs (e.g. topoisomerase-binding sites,
nuclear matrix-attachment sites).
 Use of cDNA rather than genomic DNA. (Introns thought to
contribute to stability of mRNA and may even contain enhancer
sequences essential for tissue-specific expression. Flanking DNA
may also contain regulatory sequences.)

22. Examples of Studies Utilizing
Transgenic Mice
 The Oncomouse
– c-myc oncogene + MMTV sequences  breast cancer
– Int-2 oncogene + viral promoter  prostate cancer
 To obtain abnormal expression of genes to study their
effects
– Rat growth hormone + cadmium-inducible metallothionein
promoter
– Transgenic mouse was much larger, but also suffered
complications with fertility and organ diseases

23.  To study developmentally regulated genes
 http://www.ucihs.uci.edu/anatomy/calofpix1b.html

24. More Examples of Studies
Utilizing Transgenic Mice
 “Pharm” animals (transgenic livestock)
– Bioreactors whose cells have been engineered to
synthesize marketable proteins
– DNA constructs contain desired gene and appropriate
regulatory sequences (tissue-specific promoters)
– More economical than producing desired proteins in
cell culture

25. Examples of Bioreactors
 Naked human Hb from
pigs
 Human lactoferrin in
cows’ milk
 Alpha-1-antitrypsin in
sheep
 HGH in mouse urine
(uroplakin promoters)
 Human antibodies in mice
(H and L chain tgenics 
hybridomas)
 CfTCR in goats
 Tissue plasminogen
activator (TPA) in goats
 Human antithrombin III
in goats
 Malaria antigens in goats
(vaccine)
 Alpha-glucosidase in
rabbits (Pompe’s disease

26.  http://www.bio.umass
.edu/biotech/body_bio
tech.html

27. http://www.vet.upenn.edu/catgcr/intro.htm
l

29. Transgenic Pigs for the Production
of Organs for Transplantation
 Pig organs are rejected acutely due to the
presence of human antibodies to pig antigens.
 Once human antibodies are bound to pig organs,
human complement is activated and triggers the
complement cascade and organ destruction.
 Transgenic pigs with complement inhibitors have
been produced and are gaining feasibility as a
source of xenogeneic organs for transplantation.

33. The KnockoutThe Knockout
MouseMouse

34. What is a Knockout Mouse?
 A really good-looking mouse?
 A mouse in which a very specific
endogenous gene has been altered in such
a way that interferes with normal
expression, i.e. it has been knocked out.

35. Why Produce KO Mice?
 To study effects of gene products,
biochemical pathways, alternative
(compensatory) pathways, and
developmental pathways
 To recreate human diseases in animals to
establish models to test the beneficial
effects of drugs or gene therapy.

36. Procedure for Generating
a KO Mouse
 Gene alteration in KO mice is targeted to very specific
genes.
 DNA must integrate at precise positions in the genome.
 Integration of the altered gene takes place in embryonic
stem cells ex vivo.
 Verification of exact location of integration occurs before
the ESC is introduced into blastocysts to become part of
the developing embryo.

37. Pluripotent ES Cells
 Pluripotent ES cells are undifferentiated early embryonic
cells derived from the inner cell mass of mouse
blastocysts.
 In vitro ES cells must be grown on a feeder layer of
fibroblasts to prevent them from differentiating.
 Introduction of the transgene is achieved by
electroporation of retroviral infection.
 The transgene must integrate via recombination, not
randomly.
 Cells transfected successfully can be identified prior to
injection into blastocysts.

38. Specific Gene Targeting in ES
Cells
 Gene targeting can be achieved using gene
constructs designed for homologous
recombination. This technique can be used to
either:
– Knockout functional genes to study their contribution
to different developmental or disease processes (null
mutations)
• Genes encoding β2m, MHC class I and II. CD2, Ii, TCR, Ig,
IL-4, IL-2, FcεR, TAP1/2, RAG-2,and many more (>100)!
– Replace a functional gene for a mutated/non-
functional gene to restore wild type phenotype .
• Gene encoding HGPRT in mice deficient for HGPRT (called
Lesch-Nhyan syndrome in humans).

39. DNA Constructs for
Recombination
 DNA vectors contain the gene of interest which
has been interrupted with an antibiotic resistance
gene (hygromycin resistance, or G418
resistance).
 To ensure targeted integration has occurred, the
flanking DNA contains the thymidine kinase
gene. If TK integrates (random insertion), then
the transfected cells die when grown in selective
media (gancyclovir).

41. Selection of Targeted ES Cells
 Gancyclovir resistant and G418 resistant ES cells
grow into small clumps on top of feeder cells.
 The colonies of cells can be “picked” off and
transferred to new wells (at 0.3 cells per well
seeding density) containing feeder cells.
 When sufficient numbers of cells are obtained,
they are:
– Frozen for safe storage
– Analyzed by Southern blotting or PCR to determine
nature of integration event
– Microinjected into the blastocoel cavity of blastocysts.

43.  http://www.criver.co
m/products/genetic_te
sting/dxgmon2.html

44. Dolly and theDolly and the
Advancement ofAdvancement of
Animal CloningAnimal Cloning

46. How Was Dolly Made?